Yeast Cell Permeabilization by Osmotic Shock Allows Determination of Enzymatic Activities in Situ

Crotti, Luciana B.; Drgon, Tomás; Cabib, Enrico

doi:10.1006/abio.2001.5051

Cited by 42 publications

(23 citation statements)

References 32 publications

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“…Additional incubation on ice for 5 min was followed by centrifugation and resuspension of spheroplasts in 1 ml of assay buffer (0.05 M Tris-HCl [pH 7.5], 33% glycerol). The chitin synthase assay was carried out by using the protocol described by Crotti et al (9), with slight modifications. The reaction mixtures (total volume, 50 l) contained 32 mM Tris-HCl (pH 7.5), 4.3 mM magnesium acetate, 1.1 mM ]GlcNAc) (Amersham Biosciences, Buckinghamshire, United Kingdom), 2 l of trypsin (5 mg/ml), and 20 l of spheroplasts.…”

Section: Methodsmentioning

confidence: 99%

The Antifungal Protein AFP from Aspergillus giganteus Inhibits Chitin Synthesis in Sensitive Fungi

Hagen

Marx

Ram

et al. 2007

Appl Environ Microbiol

118

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The antifungal protein AFP from Aspergillus giganteus is highly effective in restricting the growth of major human-and plant-pathogenic filamentous fungi. However, a fundamental prerequisite for the use of AFP as an antifungal drug is a complete understanding of its mode of action. In this study, we performed several analyses focusing on the assumption that the chitin biosynthesis of sensitive fungi is targeted by AFP. Here we show that the N-terminal domain of AFP (amino acids 1 to 33) is sufficient for efficient binding of AFP to chitin but is not adequate for inhibition of the growth of sensitive fungi. AFP susceptibility tests and SYTOX Green uptake experiments with class III and class V chitin synthase mutants of Fusarium oxysporum and Aspergillus oryzae showed that deletions made the fungi less sensitive to AFP and its membrane permeabilization effect. In situ chitin synthase activity assays revealed that chitin synthesis is specifically inhibited by AFP in sensitive fungi, indicating that AFP causes cell wall stress and disturbs cell integrity. Further evidence that there was AFP-induced cell wall stress was obtained by using an Aspergillus niger reporter strain in which the cell wall integrity pathway was strongly induced by AFP.

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Section: Methodsmentioning

confidence: 99%

The Antifungal Protein AFP from Aspergillus giganteus Inhibits Chitin Synthesis in Sensitive Fungi

Hagen

Marx

Ram

et al. 2007

Appl Environ Microbiol

118

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“…Strain YMS90, containing both a CHS3 and a CLA4 deletion, was obtained by transforming strain ECY46-4-1B (chs3::LEU2; Crotti et al, 2001) with a cla4::URA3 deletion cassette described by Schmidt and colleagues (Schmidt et al 2003).…”

Section: Strain Constructionmentioning

confidence: 99%

Crosslinks in the cell wall of budding yeast control morphogenesis at the mother–bud neck

Blánco

Reidy

Arroyo

et al. 2012

Journal of Cell Science

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SummaryPrevious work has shown that, in cla4D cells of budding yeast, where septin ring organization is compromised, the chitin ring at the mother-daughter neck becomes essential for prevention of neck widening and for cytokinesis. Here, we show that it is not the chitin ring per se, but its linkage to b(1-3)glucan that is required for control of neck growth. When in a cla4D background, crh1D crh2D mutants, in which the chitin ring is not connected to b(1-3)glucan, grew very slowly and showed wide and growing necks, elongated buds and swollen cells with large vacuoles. A similar behavior was elicited by inhibition of the Crh proteins. This aberrant morphology matched that of cla4D chs3D cells, which have no chitin at the neck. Thus, this is a clear case in which a specific chemical bond between two substances, chitin and glucan, is essential for the control of morphogenesis. This defines a new paradigm, in which chemistry regulates growth.

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“…Disruption of CHS2 was performed as described by Crotti et al (2001), to obtain ECY46-3-4A and ECY46-4-15C. For time-lapse microscopy of green fluorescent protein (GFP)-tagged proteins, the diploid strains DHYX100[pMS55], containing Myo1p C-terminally fused to GFP, and DHYX101[pDHR971207], containing Chs2p Nterminally fused to GFP, were used.…”

Section: Yeast Strains Constructionmentioning

confidence: 99%

The Septation Apparatus, an Autonomous System in Budding Yeast

Roh

Bowers

Schmidt

et al. 2002

MBoC

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Actomyosin ring contraction and chitin primary septum deposition are interdependent processes in cell division of budding yeast. By fusing Myo1p, as representative of the contractile ring, and Chs2p for the primary septum, to different fluorescent proteins we show herein that the two processes proceed essentially at the same location and simultaneously. Chs2p differs from Myo1p in that it reflects the changes in shape of the plasma membrane to which it is attached and in that it is packed after its action into visible endocytic vesicles for its disposal. To ascertain whether this highly coordinated system could function independently of other cell cycle events, we reexamined the septum-like structures made by the septin mutant cdc3 at various sites on the cell cortex at the nonpermissive temperature. With the fluorescent fusion proteins mentioned above, we observed that in cdc3 at 37°C both Myo1p and Chs2p colocalize at different spots of the cell cortex. A contraction of the Myo1p patch could also be detected, as well as that of a Chs2p patch, with subsequent appearance of vesicles. Furthermore, the septin Cdc12p, fused with yellow or cyan fluorescent protein, also colocalized with Myo1p and Chs2p at the aberrant locations. The formation of delocalized septa did not require nuclear division. We conclude that the septation apparatus, composed of septins, contractile ring, and the chitin synthase II system, can function at ectopic locations autonomously and independently of cell division, and that it can recruit the other elements necessary for the formation of secondary septa.

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Yeast Cell Permeabilization by Osmotic Shock Allows Determination of Enzymatic Activities in Situ

Cited by 42 publications

References 32 publications

The Antifungal Protein AFP from Aspergillus giganteus Inhibits Chitin Synthesis in Sensitive Fungi

The Antifungal Protein AFP from Aspergillus giganteus Inhibits Chitin Synthesis in Sensitive Fungi

Crosslinks in the cell wall of budding yeast control morphogenesis at the mother–bud neck

The Septation Apparatus, an Autonomous System in Budding Yeast

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